Real-time single-molecule observations of T7 Exonuclease activity in a microflow channel |
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Authors: | Shunsuke Takahashi Tomohiro Usui Shohei Kawasaki Hidefumi Miyata Hirofumi Kurita Shun-ichi Matsuura Akira Mizuno Masahiko Oshige Shinji Katsura |
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Institution: | 1. Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan;2. Department of Environmental and Life Sciences, Graduate School of Engineering, Toyohashi University of Technology, Aichi 441-8580, Japan;3. Research Center for Compact Chemical System, National Institute of Advanced Industrial Science and Technology (AIST), Miyagi 983-8551, Japan |
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Abstract: | T7 Exonuclease (T7 Exo) DNA digestion reactions were studied using direct single-molecule observations in microflow channels. DNA digestion reactions were directly observed by staining template DNA double-stranded regions with SYTOX Orange and staining single-stranded (digested) regions with a fluorescently labeled ssDNA-recognizing peptide (ssBP-488). Sequentially acquired photographs demonstrated that a double-stranded region monotonously shortened as a single-stranded region monotonously increased from the free end during a DNA digestion reaction. Furthermore, DNA digestion reactions were directly observed both under pulse-chase conditions and under continuous buffer flow conditions with T7 Exo. Under pulse-chase conditions, the double-stranded regions of λDNA monotonously shortened by a DNA digestion reaction with a single T7 Exo molecule, with an estimated average DNA digestion rate of 5.7 bases/s and a processivity of 6692 bases. Under continuous buffer flow conditions with T7 Exo, some pauses were observed during a DNA digestion reaction and double-stranded regions shortened linearly except during these pauses. The average DNA digestion rate was estimated to be 5.3 bases/s with a processivity of 5072 bases. Thus, the use of our direct single-molecule observations using a fluorescently labeled ssDNA-recognizing peptide (ssBP-488) was an effective analytic method for investigating DNA metabolic processes. |
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Keywords: | Single-molecule observation Single-stranded DNA Double-stranded DNA T7 Exonuclease |
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